Since the inception of the hybridoma technology developed by Kohler and Milstein, selection of successful hybridoma clones has been based on the selective outgrowth of hybrid cells in HAT (hypoxanthine/aminopterin/thymidine) selection medium. This prior art selection technique relies on inhibition of a synthetic pathway in the myeloma for the production of a necessary metabolite. A myeloma cell line which is deficient in hypoxanthine phosphoribosyl transferase (HPRT) is used as the fusion partner with the mammalian cell of interest. Such a mutant cell line, having no purine salvage pathway enzymes, cannot grow in HAT medium as aminopterin is an antagonist of dihydrofolate reductase and thus blocks the primary synthetic pathway. An unblocked alternative (or salvage) synthetic pathway, inherited from the mammalian fusion partner of the myeloma, exists in the hybridoma. Thus, the blockage of the synthetic pathway kills unfused myeloma cells, but not fused hybridomas, which are capable of using the necessary metabolite by an alternative pathway.
This technique, however, suffers certain drawbacks as aminopterin is inherently highly toxic to mammalian cells. Modified protocols for selecting hybridomas derived from HPRT-deficient cells have been described in which aminopterin has been replaced by the less toxic methotrexate or in which aminopterin and thymidine have been omitted in favor of azaserine. These modified selection techniques, however, still rely on inhibition of synthetic pathways in myelomas for the production of a necessary metabolite.
Unfortunately, inhibition of synthetic pathways requires the addition to the culture medium of a substance which is non-essential for cell growth. The substance can inhibit the growth of myeloma cells in ways additional to the blockage of the specified synthetic pathway and can thus inhibit the growth of fused hybridomas. Further, some of these substances have resulted in chromosomal instability. Thus, there has been an effort to develop selection media which do not rely upon inhibition of a synthetic pathway.
As noted in a paper from the laboratory of the present inventors, (Sato, J. D. et al "Cholesterol Requirement of NS-1 Mouse Myeloma Cells for growth in Serum-Free Medium", Mol. Biol. Med., 2, 121-134 (1984)), NS-1-Ag4-1 (NS-1) mouse myeloma cells, but not NS-1 hybridomas, require exogenous cholesterol for growth in serum-free medium owing to an inability to convert lanosterol to cholesterol. Based on this phenotype of cholesterol auxotrophy, it was speculated, in that paper, that cholesterol-free serum-free (CFSF) medium could be used as an alternative to HAT medium as a selective agent for NS-1 hybridomas. P3-X63-Ag8 and X63-Ag8.653 myeloma cells have the same phenotype of cholesterol auxotrophy. That paper specifically described the growth characteristics of myeloma cells in a cholesterol-free and serum-free medium supplemented with Ham's F-12 nutrient mixture. Yet, because it was unknown whether or to what extent the hybridomas would release cholesterol into the surrounding medium where it would become available for the auxotrophic myeloma cells and thus prevent selection, that paper failed to provide a reasonable expectation of success, let alone an expectation of greatly superior selection capability as compared to HAT selection.
In a later paper from the laboratory of the present inventors (J. D. Sato, T. Kawamoto, and T. Okamoto (1987), J. Exp. Med. 165: 1761-1766), the growth of myelomas in a cholesterolfree, serum-free medium lacking Ham's F-12 nutrient mixture was described, but there was no suggestion in that paper of the use of the described medium for hybridoma selection. Moreover, the growth of myelomas in the F-12 supplemented medium was not compared to the growth of myelomas in the same medium lacking F-12. At that time those skilled in the art would have expected that these growth characteristics would have been similar in each medium.